The most promising immunotherapies against cancer rely upon modulating CD8+ and various CD4+ T- cells to activate against cancers like melanoma, lymphoma, and leukemia. However, the ability for researchers to measure the secreted effector proteins per heterogeneous T-cell, which determine the cellular potency against the cancer, is limited today. This Phase 1 SBIR feasibility project proposes advancing the commercialization of a cell capture and single-cell protein analysis technology to address pharmaceutical researchers need to monitor patient T cell function and response to therapy. The prototype IsoPlexis micro- device aims to, for the first time, gather the large and necessary amounts of secreted protein data at the single immune cell level. The IsoPlexis micro-device prototype currently helps immunology researchers at Yale University and Memorial Sloan Kettering Cancer Center detect up to ten times more secreted proteins per heterogeneous immune cell versus the standard flow cytometer. Pharmaceutical firms have envisioned using this device to detect mechanism of action at the single-cell level, as well as potentially detect patient differences in immune-competency. As referenced in our support letters, immunotherapy leaders from Yale Cancer Center and California Institute of Technology are looking forward to using and participating in the development of our prototype device. IsoPlexis' micro-device has the potential to provide this necessary additional functional data to many researchers, thus advancing immunotherapy research faster and at a lower cost relative to competitor's expensive instrument technologies. The IsoPlexis device is initially targeting the T- cell analysis sub-segment of a broader $760 million market for secreted protein life science research and drug development tools. Eventually, we hope to pursue much larger scale diagnostic applications for measuring patient immune activity pre and post therapy. This Phase 1 project seeks to better determine the commercial feasibility of developing the IsoPlexis cell capture and protein analysis micro-device for mass research use, using the following specific aims. 1) Develop on-chip cell enrichment technology to facilitate single-cell multi- protein analysis of specific T-cel subsets. Here we apply DNA-encoded cell sorting to capture CD4 and CD8 for downstream analysis in our IsoPlexis micro-device prototype. 2) Develop a fully packaged micro-device to achieve automated operation and minimum commercial usability. We plan to engineer our device overcome key impediments of cell isolation, quality of quantitative detection, and detection variability. Additionally, we will pursue a pilot test for Transition to Phase II; in thi we will demonstrate minimum validity & utility through proof- of-concept comparator validation analysis of primary T-cells. Upon completion of these initial improvement goals, in Phase II IsoPlexis expects to expand product development of our cell isolation and protein analysis micro-device to provide it to the many researchers that could benefit from the tool, and to further explore diagnostic applications.